Various plants and animals exhibit biological chemiluminescence. Bioluminescence is found in microorganisms [i.e., some bacteria (mostly marine forms, e.g., Vibrio fischeri), fungi, and dinoflagellates], insects (e.g., the firefly, Photinus pyradis), some crustaceans (i.e., Cypridine hilgendorfi), jellyfish, worms and other invertebrates and even in mammals. Although the biochemical mechanism of luminescence is known to vary (i.e., the luminescence system found in bacteria is different from that found in fireflies and dinoflagellates), light production in living organisms is most frequently catalyzed by the enzyme luciferase. Bacterial luciferase is a mixed function oxidase, consisting of two different subunits each with a molecular weight of approximately 40,000 daltons.
In the bacterium Vibrio fischeri, the synthesis of the enzymes participating in the luminescenece system is regulated by a small sensory molecule, named autoinducer. During growth the autoinducer is accumulated in the growth medium. When the autoinducer reaches a critical concentration, induction of the luminescence system occurs, resulting in approximately 1000 fold increase in light production.
The preferred element of the new test is a fragment of DNA carrying the luminescence system of a luminescent bacterium, usually a marine bacterium, e.g., Vibrio fischeri.
An extracellular DNA fragment carrying the luminescence genes does not, of course, luminesce but upon transferring it to a suitable living host by transduction or transformation, the host's genetic and synthetic machinery can utilize the DNA fragment thereby causing light to be emitted. Intracellular gene segments can also be used if in the test organisms they are poorly expressed or unexpressed and only become expressed after some genetic transfer of exogenous DNA to the organism whose presence or absence is to be determined. This latter method employs conjugation, i.e., mating and genetic transfer from one cell to another.
The introduction of DNA into a bacterial cell by bacteriophage infection (transduction), by transformation or by conjugation, is usually limited with respect to the donor source: DNA transfer occurs among a group of strains of the same species or among closely related species. Some factors acting to limit such transfer include the presence of DNA restriction-modification systems in many bacterial species and/or their strains, the dependence on host factors for the replication of the introduced DNA and appropriate bacteriophage receptors in the bacterial wall upon which bacteriophage can adsorb and thereby properly inject their genetic material into the host.
Conjugation and transformation are not particularly strain specific. Some plasmids can be transferred by conjugation not only to the same species but also to related ones. There are even several plasmids which can be transferred to distant species by conjugation. Transformation is potentially even broader than conjugation since a wide range of bacteria take up extracellular DNA. Many, if not most, that are at present not known to do so may in fact be transformable under specific conditions. For example, E. coli does not normally take up DNA efficiently. However, after calcium shock this bacterium can be induced to do so at a high level of efficiency. A limiting factor in using transformation is the ability of the foreign DNA to express, replicate or integrate its genetic information into the host genome.
Unlike transformation and conjugation, transduction is quite strain or species specific. Some bacteriophages infect several species of bacteria which are usually close relatives; most infect only a particular subset of strains of a single species. By using different kinds of strains of bacteriophage which infect subsets, it is possible to arrange the strains of a bacterial species into classification schemes. This is called phage typing.
Recombinant DNA technology and molecular genetics allow the introduction of genes whose products are easily assayable. Thus, the introduction of a gene such as lac z of Escherichia coli into a bacterium can lead to expression of the introduced gene. If for example a bacteriophage is used to introduce the gene, then the course of the subsequent infection can be measured by analyzing the extent of beta-galactosidase formation. Even though the bacterial cell itself may possess a gene for this enzyme, these measurements can be performed because the endogenous level can be depressed by appropriate media and the propagation of the bacteriophage genetic material leads to multiple copies of the gene.
Although numerous exogenous genetic systems may be utilized in the. invention, the luminescent system of Vibrio fischeri is particularly useful and illustrative of our method for ascertaining the presence or absence of a given bacterial species. Only a few bacterial species contain genes allowing them to convert chemical energy to light. Most of these species are marine. The vast majority of bacterial species do not contain such systems and are dark. If the genes for light production are introduced and express themselves in a species with no such capability, then emission of light will result. The background interference in such a system is extremely low (chemical luminescence). Since very low levels of light can be detected and measured, tests based on this method should be sensitive and quantitative.